Project Summary Antibiotic resistance in bacteria is compromising our ability to treat infection. Of alarming concern is emerging carbapenem resistance amongst members of the Enterobacteriaceae (so called carbapenem-resistant Enterobacteriaceae or CRE) such as Klebsiella pneumoniae and Enterobacter spp. Heretofore, carbapenems were the class of antibiotics of last resort against multidrug-resistant pathogens. Acquisition of broadly acting enzymes that destroy these drugs has made treatment of CRE problematic at best. Alternative treatments are either absent or highly toxic. Accordingly, the CDC has classified CRE as an urgent threat. There is pressing need for novel antimicrobials and new antimicrobial target identification. Importantly, bacteria containing the same functional carbapenemase enzymes show marked differences in susceptibility to carbapenems, suggesting that carbapenem resistance is multi-factorial. In fact, some strains remain clinically susceptible both in vitro and in vivo. We therefore hypothesize that novel adjunctive antimicrobials can be found that modulate factors contributing to resistance and thereby restore the efficacy of carbapenems in non-susceptible strains. Therefore, this proposal will use a comprehensive, high-throughput screening (HTS) assay to identify novel compounds that either directly inhibit CRE and/or render CRE susceptible to carbapenems. Following identification, we aim to characterize the mechanism of action of these novel compounds and gain insight into mechanisms of carbapenem resistance and CRE antimicrobial effect. The long-term goal is to use this knowledge to develop novel effective therapies for CRE.